Summary of Study ST001892

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001191. The data can be accessed directly via it's Project DOI: 10.21228/M8RM4F This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST001892
Study Title	Small molecule signatures of mice lacking T-cell p38 alternate activation, a model for immunosuppression conditions, after exposure to total body radiation (part II)
Study Summary	Introduction Novel biodosimetry assays are needed in the event of radiological/nuclear emergencies for both immediate triage and identifying delayed effects of acute radiation exposure. Genetically engineered mouse models are used to assess how genotypic variation in the general population may affect post-irradiation classification performance. Here, we used a mouse model that lacks the T-cell receptor specific alternative p38 pathway (p38αβY323F, double knock-in [DKI] mice) to determine how attenuated autoimmune and inflammatory responses may affect dose reconstruction. Objectives To determine if deficient alternative p38 activation differentially affects biofluid metabolic signatures post-irradiation compared to wild-type (WT). Methods Untargeted global metabolomics was used to assess biofluid signatures between WT and DKI mice (8 – 10 weeks old) after exposure to total body radiation (0, 2, or 7 Gy). Urine was analyzed in the first week (1, 3, and 7 d) and serum at 1 d. Spectral features of interest were identified using the machine learning algorithm Random Forests and MetaboLyzer. Validated metabolite panels were constructed and classification performance was assessed by determining the area under the receiver operating characteristic curve (AUROC). Results A multidimensional scaling plot showed excellent separation of IR exposed groups in WT with slightly dampened responses in DKI mice. For both urine and serum, excellent sensitivity and specificity (AUROC > 0.90) was observed for 0 Gy vs. 7 Gy groups irrespective of genotype using identical metabolite panels. Similarly, excellent to fair classification (AUROC > 0.75) was observed for ≤ 2 Gy vs. 7 Gy post-irradiation mice for both genotypes, however, model performance declined (AUROC < 0.75) between genotypes post-irradiation. Conclusion Overall, these results suggest less influence of the alternative p38 activation pathway for dose reconstruction compared to other radiosensitive genotypes.
Institute	Georgetown University
Last Name	Pannkuk
First Name	Evan
Address	3970 Reservoir Rd, NW New Research Building E504
Email	elp44@georgetown.edu
Phone	2026875650
Submit Date	2021-07-23
Raw Data Available	Yes
Raw Data File Type(s)	raw(Waters)
Analysis Type Detail	LC-MS
Release Date	2022-07-06
Release Version	1

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Project:

Project ID:	PR001191
Project DOI:	doi: 10.21228/M8RM4F
Project Title:	Small molecule signatures of mice lacking T-cell p38 alternate activation, a model for immunosuppression conditions, after exposure to total body radiation
Project Summary:	Introduction Novel biodosimetry assays are needed in the event of radiological/nuclear emergencies for both immediate triage and identifying delayed effects of acute radiation exposure. Genetically engineered mouse models are used to assess how genotypic variation in the general population may affect post-irradiation classification performance. Here, we used a mouse model that lacks the T-cell receptor specific alternative p38 pathway (p38αβY323F, double knock-in [DKI] mice) to determine how attenuated autoimmune and inflammatory responses may affect dose reconstruction. Objectives To determine if deficient alternative p38 activation differentially affects biofluid metabolic signatures post-irradiation compared to wild-type (WT). Methods Untargeted global metabolomics was used to assess biofluid signatures between WT and DKI mice (8 – 10 weeks old) after exposure to total body radiation (0, 2, or 7 Gy). Urine was analyzed in the first week (1, 3, and 7 d) and serum at 1 d. Spectral features of interest were identified using the machine learning algorithm Random Forests and MetaboLyzer. Validated metabolite panels were constructed and classification performance was assessed by determining the area under the receiver operating characteristic curve (AUROC). Results A multidimensional scaling plot showed excellent separation of IR exposed groups in WT with slightly dampened responses in DKI mice. For both urine and serum, excellent sensitivity and specificity (AUROC > 0.90) was observed for 0 Gy vs. 7 Gy groups irrespective of genotype using identical metabolite panels. Similarly, excellent to fair classification (AUROC > 0.75) was observed for ≤ 2 Gy vs. 7 Gy post-irradiation mice for both genotypes, however, model performance declined (AUROC < 0.75) between genotypes post-irradiation. Conclusion Overall, these results suggest less influence of the alternative p38 activation pathway for dose reconstruction compared to other radiosensitive genotypes.
Institute:	Georgetown University
Last Name:	Pannkuk
First Name:	Evan
Address:	3970 Reservoir Rd, NW New Research Building E504
Email:	elp44@georgetown.edu
Phone:	2026875650
Publications:	https://meridian.allenpress.com/radiation-research/article-abstract/197/6/613/478727/Small-Molecule-Signatures-of-Mice-Lacking-T-cell

Subject:

Subject ID:	SU001970
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Genotype_irradiation
SA175953	26	dki_2Gy
SA175954	24	dki_2Gy
SA175955	27	dki_2Gy
SA175956	29	dki_2Gy
SA175957	31	dki_2Gy
SA175958	30	dki_2Gy
SA175959	25	dki_2Gy
SA175960	28	dki_2Gy
SA175961	33	dki_7Gy
SA175962	37	dki_7Gy
SA175963	36	dki_7Gy
SA175964	35	dki_7Gy
SA175965	34	dki_7Gy
SA175966	32	dki_7Gy
SA175967	42	dki_7Gy
SA175968	43	dki_7Gy
SA175969	44	dki_7Gy
SA175970	38	dki_7Gy
SA175971	41	dki_7Gy
SA175972	40	dki_7Gy
SA175973	39	dki_7Gy
SA175974	23	dki_sham
SA175975	17	dki_sham
SA175976	16	dki_sham
SA175977	18	dki_sham
SA175978	19	dki_sham
SA175979	21	dki_sham
SA175980	20	dki_sham
SA175981	22	dki_sham
SA175938	6	WT_2Gy
SA175939	8	WT_2Gy
SA175940	10	WT_2Gy
SA175941	7	WT_2Gy
SA175942	9	WT_2Gy
SA175943	12	WT_7Gy
SA175944	14	WT_7Gy
SA175945	13	WT_7Gy
SA175946	11	WT_7Gy
SA175947	15	WT_7Gy
SA175948	2	WT_sham
SA175949	1	WT_sham
SA175950	5	WT_sham
SA175951	3	WT_sham
SA175952	4	WT_sham

Showing results 1 to 44 of 44

Showing results 1 to 44 of 44

Collection:

Collection ID:	CO001963
Collection Summary:	Serum was collected after irradiation
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR001982
Treatment Summary:	WT C57Bl/6 mice (C57BL/6NCrl strain code #027) were obtained from Charles River Laboratories (Frederick, MD) and DKI mice were kindly provided by the Laboratory of Immune Cell Biology, National Cancer Institute (P.I. Jonathan D. Ashwell, M.D.) (Jirmanova et al. 2011). Animals were bred/irradiated (12 h light / 12 h dark cycle conditions) at Georgetown University and water and food (PicoLab Rodent Diet 20 #5053) were provided ad libitum according to Georgetown University Institutional Animal Care and Use Committee (GUACUC) protocols (2016-1152). Before irradiation and biofluid collection the mice were acclimated to metabolic cages for 24 h. Male mice that were 8 – 10 weeks old were exposed to a total body ionization (TBI) x-ray dose (~1.67 Gy/min; X-Rad 320, Precision X-Ray Inc, Branford, CT; filter, 0.75 mm tin/ 0.25 mm copper/1.5 mm aluminum) of 0, 2, or 7 Gy. All urine samples were collected over a 24 h period in a metabolic cage pre-irradiation and at days 1, 3, and 7 d post-irradiation (Figure S1). Blood for metabolomics was collected at 1 d via cheek bleed from the submandibular vein and serum was separated in a BD microtainer serum separator tube and centrifuged for 10 min (10,000 x g, 4°C). Serum samples from sham-irradiated mice were used as a control (Figure S1). All biofluids were flash frozen and stored at -80°C until further use. Seven days post-irradiation, blood was collected in a dipotassium EDTA Tube (BD Cat #365974) via the facial vein from each animal and subjected to a complete blood count by VRL Diagnostics (Gaithersburg, MD, http://www.vrlsat.com/) (Figure S2).

Treatment ID:

TR001982

Treatment Summary:

WT C57Bl/6 mice (C57BL/6NCrl strain code #027) were obtained from Charles River Laboratories (Frederick, MD) and DKI mice were kindly provided by the Laboratory of Immune Cell Biology, National Cancer Institute (P.I. Jonathan D. Ashwell, M.D.) (Jirmanova et al. 2011). Animals were bred/irradiated (12 h light / 12 h dark cycle conditions) at Georgetown University and water and food (PicoLab Rodent Diet 20 #5053) were provided ad libitum according to Georgetown University Institutional Animal Care and Use Committee (GUACUC) protocols (2016-1152). Before irradiation and biofluid collection the mice were acclimated to metabolic cages for 24 h. Male mice that were 8 – 10 weeks old were exposed to a total body ionization (TBI) x-ray dose (~1.67 Gy/min; X-Rad 320, Precision X-Ray Inc, Branford, CT; filter, 0.75 mm tin/ 0.25 mm copper/1.5 mm aluminum) of 0, 2, or 7 Gy. All urine samples were collected over a 24 h period in a metabolic cage pre-irradiation and at days 1, 3, and 7 d post-irradiation (Figure S1). Blood for metabolomics was collected at 1 d via cheek bleed from the submandibular vein and serum was separated in a BD microtainer serum separator tube and centrifuged for 10 min (10,000 x g, 4°C). Serum samples from sham-irradiated mice were used as a control (Figure S1). All biofluids were flash frozen and stored at -80°C until further use. Seven days post-irradiation, blood was collected in a dipotassium EDTA Tube (BD Cat #365974) via the facial vein from each animal and subjected to a complete blood count by VRL Diagnostics (Gaithersburg, MD, http://www.vrlsat.com/) (Figure S2).

Sample Preparation:

Sampleprep ID:	SP001976
Sampleprep Summary:	Biofluids were prepared as previously described (Pannkuk et al. 2018;2020). Urine (20 μl) was deproteinated with 50% acetonitrile (80 μl) containing internal standards (2 μM debrisoquine sulfate, 30 μM 4-nitrobenzoic acid), incubated on ice for 10 min, vortexed for 30 seconds, and centrifuged for 10 min (10,000 x g, 4°C). Serum (5 μl) was prepared as above but was deproteinated with 66% acetonitrile (195 μl). A quality control (QC) sample was prepared by mixing 1 μl from each sample and prepared as above.
Processing Storage Conditions:	-80℃

Combined analysis:

Analysis ID	AN003072	AN003073
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Waters Acquity	Waters Acquity
Column	Waters Acquity BEH C18 (50 x 2.1mm,1.7um)	Waters Acquity BEH C18 (50 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Waters Synapt G2	Waters Synapt G2
Ion Mode	POSITIVE	NEGATIVE
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH002273
Chromatography Summary:	Mobile phases consisted of the following: solvent A (water/0.1% formic acid [FA]), solvent B (ACN/0.1% FA), solvent C (isopropanol [IPA]/ACN (90:10)/0.1% FA). The gradient for urine was (solvent A and B) 4.0 min 5% B, 4.0 min 20% B, 5.1 min 95% B, and 1.9 min 5% B at a flow rate of 0.5 ml/min. The gradient for serum was (solvent A, B, and C) 4.0 min 98:2 A:B, 4.0 min 40:60 A:B, 1.5 min 2:98 A:B, 2.0 min 2:98 A:C, 0.5 min 50:50 A:C, and 1.0 min 98:2 A:B at a flow rate of 0.5 ml/min.
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C18 (50 x 2.1mm,1.7um)
Flow Gradient:	The gradient for urine was (solvent A and B) 4.0 min 5% B, 4.0 min 20% B, 5.1 min 95% B, and 1.9 min 5% B at a flow rate of 0.5 ml/min. The gradient for serum was (solvent A, B, and C) 4.0 min 98:2 A:B, 4.0 min 40:60 A:B, 1.5 min 2:98 A:B, 2.0 min 2:98 A:C, 0.5 min 50:50 A:C, and 1.0 min 98:2 A:B at a flow rate of 0.5 ml/min.
Flow Rate:	0.5 ml/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	solvent B:100% acetonitrile; 0.1% formic acid solvent C:90% isopropanol/10% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002859
Analysis ID:	AN003072
Instrument Name:	Waters Synapt G2
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Negative and positive electrospray ionization (ESI) data-independent modes were used for data acquisition with leucine enkephalin ([M+H]+ = 556.2771, [M-H]- = 554.2615) as Lock-Spray®. Operating conditions for ESI were: capillary voltage 2.75 kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow 1000 L/Hr.
Ion Mode:	POSITIVE

MS ID:	MS002860
Analysis ID:	AN003073
Instrument Name:	Waters Synapt G2
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Negative and positive electrospray ionization (ESI) data-independent modes were used for data acquisition with leucine enkephalin ([M+H]+ = 556.2771, [M-H]- = 554.2615) as Lock-Spray®. Operating conditions for ESI were: capillary voltage 2.75 kV, cone voltage 30 V, desolvation temperature 500°C, desolvation gas flow 1000 L/Hr.
Ion Mode:	NEGATIVE